Hi. I'm using the pDisplay vector from Invitrogen to express in the membrane surface a cytosolic domain of a protein.
The problem I have is that the protein is not displaying in the surface, I already sequenced the plasmid and it have no mutations and my construction is in frame with PDGFR-Transmembrane domain. Also, I checked the expression of the protein and it is expressing but it seems that the protein stays in the traffic vesicles but never reach the cell membrane.
If someone has an idea of what is happening or has any suggestion in displaying proteins in the surface of cell membrane, please leave a comment.
Do you have a control protein that does reach the cell membrane in this way?
It could have an internal sorting motif that causes it to stay in non-secretory vesicles. For example, a lysosomal address tag, or a "KDEL" ER retention signal?
And finally try dropping some calcium ionophore. It can mobilize vesicles to secrete themselves. (But it might mess with your experiment, depending on what you're trying to do...)
To add to John's reply: the absence of glycosylation or other protein maturation events can also dramatically change your proteins localization.
Are there any post-translational modifications that are required for function in the literature? Do they seem to be related to localization? Is this an ECD of a species that is (significantly) different than the cell line? Why are you unable to use the WT full length protein?
your protein might be having a signal motif for targeting to some other parts of the cell like lysosomes. I guess your protein is codon optimized to expressin in human cells. Sometimes, larger proteins and excessive expression of protein have difficulties in expressing protein on the cell surface. The best way to rule out possibilities is to have a control protein cloned under PDGFR which can come to cell surface. If you have any membrane protein construct with you, try cloning the transmembrane part of the membrane protein in the place of PDGFR and try for cell surface expression, sometimes it may work.
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